Detection of Endogenous Superoxide Production in M. smegmatis

Superoxide production in M. smegmatis was detected by the following modified HPLC-based method, described earlier (Laurindo et al., 2008; Zielonka et al., 2008, 2009). Briefly, ∼2.2 × 10⁸ cells/mL of aerobically growing M. smegmatis culture was washed and re-suspended in 1 mL of M. phlei medium (Khan et al., 2008) containing diethylenetriaminepentaacetic acid (DTPA) and incubated with DHE at a final concentration of 50 μM at 37°C for 90 min. After incubation, the cell pellet was obtained by centrifugation at 10,000 rpm for 10 min at 4°C, washed twice with M. phlei medium, and re-suspended in 500 μL of the same medium containing 1–2% (v/v) of Triton X100. After mixing, an equal volume of acidified methanol [with 1% formic acid (v/v)] was added and the cell suspension mix was kept on ice for 90 min. The supernatant was collected after centrifugation at 15,000 rpm for 45 min at 4°C and filtered through a 0.2-μm membrane filter (Acrodisc 13 mm syringe filters, Pall Corporation). The filtrate was then analyzed using HPLC (Binary pump-1525, Fluorescence detector-2475, UV detector-2489, Sampler-2707, Waters, India). The chromatographic separation was performed on a C18 reverse phase column (Kinetex 5 μm C18 100A, 250 mm × 4.60 mm from Phenomenex, India). A gradient of solutions A [1% (v/v) formic acid in water] and B [1% (v/v) formic acid in acetonitrile] was used as the mobile phase at a flow rate of 0.4 mL/min. Chromatographic runs were started with 100% solution A, decreased linearly to 60% solution A during the first 8 min, and then kept constant at isocratic condition for a further 12 min. In the next 1 min, the gradient was set at 100% solution B, kept constant for a further 4 min, and equilibrated again with solution A for the next run. The 2-hydroxyethidium fluorescence was monitored by a fluorescence detector (excitation 480 nm, emission 580 nm). The area under the curve of 2-hydroxyethidium and ethidium were considered and their concentrations were calculated based on a standard plot.

Superoxide production in the crude membrane was assessed by the method described above. Briefly, 15 μg of the crude membrane protein was added to 500 μL of M. phlei medium containing 100 μM DTPA and 1 mM NADH. Freshly prepared DHE was added at a final concentration of 25 μM and the mix was incubated at 37°C, following which HPLC samples were prepared as described earlier in this section.